Process and device for optically measuring a point on a sample with high local resolution

ABSTRACT

The invention relates to a device for the optical measurement of a point (7) on a sample (8) with high-local resolution, with a light source (1) to emit a beam (16) suitable for exciting an energy state in the sample (8), and a detector (9) to detect the emitted light. The lateral resolution of the device is improved in that there is a stimulation light beam (17) from the exciting light source (1) to generate stimulated emission at the point (7) on the sample (8) excited by the light beam (16), in which the exciting light beam (16) and the stimulation light beam (17) are arranged in such a way that their intensity distributions partly overlap in the focal region.

The invention concerns a device for optically measuring a point on asample with high local resolution, with a light source to send out aexciting light beam suitable for exciting an energy state on the sample,a lens for focusing the exciting light beam on the points, the samplewhich can be arranged in the focal area of the lens, a separating devicefor separating out the emission light spontaneously emitted from thesample based on the excitation of the energy state and a detector fordetecting the emission light.

The invention also concerns a process for optically measuring a point ona sample with high local resolution, in which an exciting light beam isfocused on the point to be measured by means of a lens and excites theenergy state there, and in which the emission light spontaneouslyemitted by the point, based on the excitation of the energy state, isseparated out and detected.

FIELD OF THE INVENTION

The invention relates generally to an optical measurement device andmore specifically to a device which performs optical measurements of asample with high local resolution.

BACKGROUND

Such a device and such a process are known from practice. They haveapplications, for example, in microscopes, particularly rastermicroscopes. With a raster microscope, individual points on a sample arescanned and measured. In this way, the sample can be measuredthree-dimensionally. Luminescent, particularly fluorescent orphosphorescent samples, or samples with corresponding dyes, are used.

In such a device and such a process, it is desirable to achieve goodlocal resolution. The local resolution is given by the spatial expansionof the so-called effective point-imaging function. This is aplace-dependent function, which quantifies the probability with which aphoton will be spontaneously emitted from a certain point in the focalrange. It is identical to the spatial distribution of the probabilitythat the energy state is excited at a certain point in the focal range.In conventional fluorescent microscopes,the effective point-imagingfunction of the lens at the wavelength of the excitation light, whichgives the distribution of the intensity of the excitation light in thefocal range of the lens and quantifies the probability with which anexposure photon will be met at a certain point in the focal range from aquantum-mechanics standpoint. In a raster microscope, the rasterdivision is limited by the local resolution. With better localresolution, a finer division can therefore be selected, with which abetter resolution of the reconstructed image can be achieved.

It is known that in a raster microscope, the resolution can be improvedby having the light detected by the sample imaged on the point detector,which is arranged in one of the planes conjugated to the focal plane ofthe lens. Such an arrangement is called confocal. The better resolutionis caused by the fact that two point-imaging functions determine theimage in the confocal raster microscope: The effective point-imagingfunction and the detection-imaging function, which describes the imageof the light to be detected that is emitted by the sample in the pointdetector and quantifies the probability with which a photo emitted fromthe focal range goes into the point detector from a quantum-mechanicsstandpoint. Since both exposure and detection must take place, thepoint-imaging function of a confocal raster microscope is the product ofboth probability distributions, i.e., from the effective point-imagingfunction and the detection-point-imaging function. This leads to aclearly narrower main maximum of the confocal point-imaging functioncompared to a microscope not arranged confocally. This corresponds to ahigher resolution of the confocal microscope and brings aboutdiscrimination of all points that are not in the direct vicinity of thefocus. The latter is the prerequisite for making three-dimensionalimages in the raster process.

SUMMARY

The task of the invention is to improve the generic device and thegeneric process in such a way that greater local resolution is achieved.

Regarding the device, this task is solved by the invention with asimulation light beam coming from the light source to produce stimulatedemission of the sample excited by the exciting light beam in the point,wherein the exciting light beam and the stimulating light beam arearranged in such a way that their intensity distributions partiallyoverlap in the focal range.

Regarding the process, the invention solves the task by having thesample excited by the exciting light beam in the point induced tosimulated emission by a simulation light beam, wherein the intensitydistributions of the exciting light beam and the stimulating light beampartially overlap in the focal range of the lens.

The stimulated emission induced by the stimulating light beam of thesample excited by the exciting light beam in the range covered by theintensity distributions of the excitation light and the stimulatinglight in the focal range of the lens make the excited energy state inthe range covered calm down so it can no longer contribute to thespontaneously emitted radiation to be detected. The effectivepoint-imaging function, which in the normal fluorescence microscope isidentical to the point-image function of the lens at the wavelength ofthe excitation light, is thereby made narrower. This corresponds toincreased spatial resolution. The improvement in the local resolutiondepends on the type of coverage of the intensity distributions. Both alateral and an axial improvement in the local resolution can beachieved.

According to one version of the invention, it is advantageous if thesample is arranged on a positioning table, with which a mechanicalraster movement can be carried out, at least in the direction of theoptical axis. The device then corresponds to a raster microscope inwhich the sample can be scanned, at least along the optical axis. Inthis case, an improvement in local resolution in the axial direction isespecially advantageous, since then better resolution can be achieved inthat direction through finer rastering. Another advantage can beachieved if there is a beam-raster device for controlled scanning of thesample, with the exciting light beam and the stimulating light beambetween the light source and the lens. The device is used as a rastermicroscope in which the sample can be scanned laterally orthree-dimensionally. In such a raster microscope, better localresolution can also be achieved in the lateral direction by making therastering smaller.

It is also advantageous if the stimulating light beam is moved laterallyin the focal plane with regard to the exciting light beam. Thisarrangement makes the effective point-imaging function of the devicenarrower in the lateral direction. It can also be helpful if thestimulating light beam is moved along the optical axis in relation tothe exciting light beam. This then improves the local resolution of thedevice in the axial direction.

Advantageously, there can also be at least one other stimulating lightbeam coming from the light source whose intensity distribution in thefocal range of the lens is different from the intensity distribution ofthe other stimulating beams. In this arrangement, the intensitydistributions of the additional stimulating beams also overlap theintensity distribution of the exciting light beam in the focal range ofthe lens, which again narrows the effective point-imaging function ofthe device. The type of narrowing of the effective point-imagingfunction can be chosen by the spatial arrangement of the stimulatinglight beams. Advantageously, the stimulating light beams can bespatially arranged symmetrically in relation to the exciting light beam.For example, the stimulating light beams can be arranged so that theyrun through a circular ring concentric to the exciting light beam. Here,the stimulating beams can be the same distance from one another. Thatway, the main maximum of the intensity distribution of the excitinglight beam is narrowed evenly, so to speak, from several sides. Otherarrangements of the stimulating light beams are also possible, and theprecise choice of arrangement is left to the expert.

According to one advantageous version of the invention, the light sourcecan include a laser, which emits portions of light of differentwavelengths. The light of one wavelength is then used as the excitationlight. The wavelength of the light is selected so that the energy statusof the sample can thus be excited. The portion of light with the otherwavelength is chosen for the stimulating light. The wavelength must bechosen so that the sample in the excited state can be calmed down viastimulated emission. Normally, the wavelengths necessary for theexcitation light and the simulation light are different from oneanother. In the event that these wavelengths are the same, of course onecan simply use a laser that only emits one wavelength.

It can also be advantageous if the light source includes at least twolasers, which emit light of different wavelengths. Then one laser isused to produce the excitation light and the other laser(s) to producethe stimulating light. Several stimulating light beams can be producedeither with a laser, which is possible with a suitable filter or anappropriate array of mirrors, or several lasers can be used to produceone or more stimulating beams each. The use of lasers as a light sourcealso has the advantage that light beams that can be highly localizedspatially with high intensity are available.

More advantageously, a continuous-wave laser can be provided, whichsends out excitation light. Using a continuous wave laser makes thearrangement less expensive. At least one laser can be provided thatsends out a light pulse in a time sequence. More advantageously, a laserthat sends out light pulses in a time sequence produces the stimulatinglight.

One advantageous arrangement consists of a continuous-wave laser toproduce the excitation light and at least one laser that beams out lightpulses in a time sequence to produce the stimulating light. The timewithin which the luminescence light should be detected by the detectoris determined by the pulse length of the stimulating light. Here it isadvantageous if the pulse length of the laser that emits the stimulatinglight is 10⁻¹⁰ to 10⁻⁵.

According to another advantageous arrangement, both the excitation lightand the stimulating light are produced by lasers that emit light pulsesin time sequence. In this case, the pulse length of the excitation lightand the stimulating light should be smaller than the characteristictimes for the spontaneous emission of the sample in the excited energystate. The pulse length of the stimulating light should be longer thanthe characteristic time for a dissociation process of the end state inwhich after the energy state is quieted down by stimulating emission,the sample is in a basic state that is even lower. From the latter, thesample is typically excited in the energy state. The pulse length of theexcitation light is advantageously 10⁻¹⁵ to 10⁻⁹ s; the pulse length ofthe stimulating light is advantageously 10⁻¹² to 10⁻⁹ s.

Advantageously, the laser can send out a light beam with a Gaussianintensity distribution to produce the stimulating light. That way, aGaussian spatial intensity distribution is achieved in the focal planeas well. Such an intensity distribution has the advantage that it has noauxiliary maxima that could make the resolution worse. This isespecially advantageous for the stimulating beams, since they can thenbe overlapped with the excitation beam so that they laterally overlapthe main maximum of the intensity distribution of the excitation beam.In this case, any lateral maxima in the intensity distribution of theexciting light beam are eliminated because of the effect of thestimulating light beams in the effective point-imaging function. Thestimulating light beams of the intensity distribution of the excitationbeam can be overlapped from outside without any new auxiliary maximumbeing created in the effective point-imaging function. In this case, aclear narrowing of the main maximum of the effective point-imagingfunction is achieved without any auxiliary maxima occurring.

It is also favorable if the light source for producing the stimulatedemission is high intensity, so that there is a nonlinear connectionbetween that intensity and the occupation of the energy state of thesample. That way, with the stimulating light beam, the excited energystate in the range covered by the intensity distribution of the excitingand stimulating light can be quieted down by stimulated emission in away that is very sharply limited spatially, so that the effectivepoint-imaging function is also very sharply limited spatially, and atthe same time a reduction in intensity of the overall luminescence isminimized.

According to one advantageous example of embodiment of the invention,the separating device has a time-control device, with which the detectorcan be turned on only directly after the pulse of the stimulating lightdies. In this arrangement, if lasers that send out light pulses in atime sequence are used to produce both the excitation light and theseparation light, the time-control device can also control the lasers insuch a way that a simulation light pulse is emitted as soon as anexcitation light pulse has died. The detector can then be activated withthe same time-control device after the pulse of the stimulating lightdies. The preferred pulse lengths of the laser were already mentionedabove. A simple, clean separation of the emission light of the sample ispossible even when the excitation light has the same wavelength as theemission light. The design of the device is mechanically simple, sinceno other filter elements have to be used.

According to another advantageous example of embodiment of theinvention, the separation device can include a polarization elementconnected on the input side of the lens to polarize the stimulatinglight and a polarization element connected on the output side of thelens to polarize the light going to the detector with a conductingdirection orthogonal to the polarization element connected to the lenson the input side. Connected on the input or output side here means bothspatially and also in the direction in which the light runs before orafter the lens. That way, it is possible to separate the emission lightfrom the stimulating light reliably when the wavelengths are the same. Apolarization element to polarize the excitation light can also beconnected to the lens on the input side and another polarization elementconnected to the lens on the output side, which has a conductingdirection orthogonal to the polarization element to polarize the lightgoing to the detector. Thus, the light emitted by the sample can also beseparated from the excitation light. It is also advantageous if theseparation device has at least one wavelength filter. With thewavelength filter, the light emitted by the sample can be separated fromthe excitation light, if there are different wavelengths. A wavelengthfilter is connected to the lens on the output side. Color filters,dichroitic filters, monochromators, prisms, etc. can be used aswavelength filters. The separation device can also have a dichroiticmirror. The mirror is then arranged between the light source and thelens so that the light emitted from the sample is deflected by thedichroitic mirror, if it has a certain wavelength, and into thedetector.

According to another advantageous version of the invention, the detectorcan be a point detector. A focusing element and a filter can beconnected to the detector on the input side, wherein the filter isarranged in a plane optically conjugated to the focal plane of the lens.

The filter is, for example, an aperture, wherein its diameter ispreferably so large that its image in the sample range is on themagnitude of the expansion of the effective point-imaging function atthe wavelength of light to be detected. The point-imaging function ofthe device comes from the product of the effective point-imagingfunction and the detection-point-imaging function. Based on the pointdetector, an additional narrowing of the main maximum of thepoint-imaging function of the device and hence another improvement inresolution is thus achieved.

Another improvement in the local resolution of the device can beachieved by arranging a filter element permeable for the wavelength ofthe stimulating light between the light source and the lens that has animpermeable middle area and a permeable outer area for the wavelength ofthe excitation light. Such a filter element moves light from the mainmaximum into the bending auxiliary maxima in the intensity distributionof the excitation light in the focal range, wherein the main maximum ismade clearly narrower. This leads to another narrowing of the effectivepoint-imaging function. The increase in the intensity of the auxiliarymaxima of the intensity distribution of the excitation light is, in thiscase, not disturbing, since they are suppressed because of the intensitydistribution of the simulation light in the effective point-imagingfunction, since the intensity distributions partially overlap.

DESCRIPTION OF THE DRAWINGS

The invention will be described in greater detail below using thedrawings.

FIG. 1 shows a schematic drawing of one example of embodiment of thedevice in the invention.

FIG. 2 shows an example of the intensity distribution of the excitationlight and the intensity distributions of the stimulating light in thefocal plane of the lens of the device in the invention and

FIG. 3 shows the effective point-imaging function in the focal plane ofthe lens of the device in the invention.

DETAILED DESCRIPTION

FIG. 1 shows, the arrangement of a raster microscope as an example ofembodiment of the device in the invention. The raster microscopeincludes a light source 1 with a laser 2 to emit exciting light and alaser 3 to emit stimulating light. There are also dichroitic mirrors 4and 5 and a lens 6, with which the exciting light and stimulating lightcoming from lasers 2 and 3 are deflected or focused on one point 7 ofthe sample 8. The point 7 has a local expansion which is an expansion ofthe surface here. A detector 9 is arranged to detect the emission lightemitted by the sample 8 which is separated from the excitation light 18with the dichroitic mirror 5. The sample is arranged on a positioningtable 10. Between the light source 1 and the lens 6 is a beam-rasterdevice 11 for controlled scanning of the sample 8 with the excitinglight and the stimulating light. The light source 1 includes, inaddition to the lasers 2 and 3, lenses 12 and 13 and a filter 14. Withthe lenses 12, the laser beam coming from the laser 2 is focused on thefilter 14. The lens 13 is used to adjust the divergence of the excitinglight beam and the stimulating light beams, so that they can be focusedin the same plane using the lens 6. Apertures are usually used asfilters. Behind the laser 3, there is a beam splitter 23 and a mirror 24for dividing up the beam coming from the laser 3 into two stimulatinglight beams 17. The arrangement is selected so that the exciting lightbeam and the stimulating light beams meet on the mirror 4 in such a waythat after deflection at the mirror 5 and passage through the lens 6,the intensity distributions of the beams partially overlap in the focalrange of the lens 6.

In the example of embodiment shown, there are two stimulating lightbeams. However, one or more stimulating light beams may also be used.For this, either other lasers with beam paths similar to the beam pathof the laser 3 can be used, wherein the beams are deflected in asuitable way to the mirror 4 and from there via the mirror 5 and thelens 6 to the point 7. But a laser 3 can also be used, as shown, and thestimulating light beam coming from the laser 3 can be broken intoindividual stimulating light beams using other beam splitters. It isimportant that the stimulating light beams all be arranged in such a waythat their intensity distributions in the focal range of the lens 6partially overlap with the intensity distribution of the exciting lightbeam. The laser 3 and the accompanying beam elements, not shown, such asbeam splitters, lenses, etc. must be arranged so that the desired,predetermined arrangement of intensity distributions occurs in the focalrange of the lens 6. For example, various stimulating light beams can bearranged on a circular ring through whose center point the excitinglight beam 16 coming from the laser 2 runs.

In point 7, the energy state of the sample 8 is excited with theexciting light beam 16 that hits it there. The wavelength of theexciting light is chosen so it is suitable for exciting this energystate. Due to the impact of the stimulating light beam 17 coming fromthe laser 3, the energy state of the sample 8 excited with the excitinglight is quieted via stimulated emission into a deeper state. For this,the laser 3 can emit the stimulating light as light pulses in a timesequence. The emission light spontaneously emitted by the sample 8 isdeflected by the lens 6 and the mirror 5 into the detector 9 anddetected there. In the drawing shown, the emission light is separatedfrom the excitation light 16 by the dichroitic mirror 6 for detection inthe detector 9. This is possible because as a rule the excitation light16 has another wavelength than the emission light 18. For a furtherimprovement in the selection of the emission light 18 based on itswavelength, a color filter 19 is arranged in front of the detector 9.

A polarizer 20 connected on the input side to the lens 6 to polarize thestimulating light 17 and a polarizer 21 connected on the output side tothe lens 6 to polarize the light going to the detector are provided. Thepolarizers 20 and 21 have a conducting direction orthogonal to oneanother. With the polarizers 20 and 21, the emission light coming fromthe sample 6 can be separated from the stimulating light. The polarizers20, 21 are necessary, since the stimulating light and the emission lighthave the same wavelengths and thus cannot be separated from one anotherby color filters. Moreover, an aperture 15 is connected on the inputside to the color filter 19 and the polarizer 21, and it is in a planeoptically conjugated to the focal plane of the lens. Alternately, theemission light coming from the sample can be separated from thestimulating light or the exciting light by a time-control device, notshown. This is possible when the laser 3 sends out stimulating lightpulses in a time sequence and the laser 2 exciting light pulses in atime sequence. The time-control device must be turned on to the detectorright after a pulse of the stimulating light dies. That way, theemission light can be separated from the stimulating light easily andreliably. The filter 19 and polarizers 20, 21 can be also arranged asshown in FIG. 1, if need be.

The beam raster device 11 is connected on the input side to a lens 22,with which the exciting light beam 16 and the stimulating light beams 17are focused in the beam raster device 11. With the beam raster device11, the exciting light beam 16 and the stimulating light beam 17 arecontrolled so that they scan the points 7, 7', . . . of the sample 8 ina desired sequence. In each of the points 7, 7', the measurementdescribed above is taken.

FIG. 2 shows the intensity distribution 25 of the exciting light beamand the intensity distributions 26 of two stimulating light beams 17.The intensity distribution 25 of the exciting light beam 16 has a mainmaximum and symmetrical auxiliary maxima in the lateral direction. Theintensity distribution 26 of the stimulating beams 17 are Gaussian. Themaxima of the Gauss distributions of the stimulating light are staggeredlaterally in relation to the maximum of the intensity distribution 25 ofthe excitation light. A symmetrical arrangement is chosen in which thetwo stimulating light beams are moved in the opposite direction at thesame distance in relation to the central axis by the intensitydistribution 25 of the exciting light. The intensity of the stimulatinglight is clearly greater than the intensity of the excitation light. Theintensity of the stimulating light beam is chosen so that there is anonlinear connection between that intensity and the occupation of theenergy state of the sample.

FIG. 3 shows the effective point-imaging function in the focal plane ofthe lens, in which the intensity distributions in FIG. 2 go. Theeffective point-imaging function determines the local resolution of araster microscope. As can be seen in the figure, the resulting effectivepoint-imaging function has a maximum whose half-width value is clearlynarrower than the half-width value of the intensity distribution of theexciting light, see FIG. 2. Also, through the overall effect of theintensity distributions shown in FIG. 2, the auxiliary maxima containedin the excitation light are eliminated. Because of the interaction ofthe exciting light beam and the stimulating light beams, one thusobtains an effective point-imaging function with a substantialimprovement in lateral resolution, since both the half-width value ofthe effective point-imaging function is substantially reduced, and alsothe auxiliary maxima contained in the intensity distribution of theexciting light are eliminated, compared with the effective point-imagingfunction of a conventional fluorescence microscope, which is identicalto the intensity distribution of the excitation light in the focal rangeof the lens. Because of the computational results, with the process inthe invention, the lateral resolution of a raster microscope can beimproved by a factor of roughly 5. The curves shown in FIGS. 2 and 3show this in principle, but not to scale.

The process in the invention will be described below using FIG. 1. Thesample 8 is put into a certain position on the optical axis A in theraster microscope shown with the positioning table 10. The lasers 2, 3and the lenses 12, 13, the filter 14, the beam splitter 23 and themirror 24 are arranged so that the stimulating light beams 17 and theexciting light beam 16 are deflected by the mirror 5 and the lens 6 to aselected sample point 7. The stimulating light beams 17 are aligned sothat their intensity distributions overlap in the focal range of thelens 6 with the intensity distribution of the exciting light beam 16 inthe way desired. The filter 19 and the polarizers 20, 21 are arranged sothat the emission light emitted by the sample at the point 7 isseparated from the exciting light and the separation light and isdeflected into the detector 9 and detected there. After thismeasurement, a new point 7'is selected. For this, the exciting lightbeam 16 and the stimulating light beam 17 are deflected to the point 7'.There they are measured the same way as in point 7. After that, theexciting light beam 16 and the stimulating light beams 17 are deflectedby the beam-raster device 11 to another point, until the sample 8 hasbeen scanned and measured in the desired range in the lateral direction.Then the positioning table 10 is moved in the direction of the opticalaxis A. In that position of the sample 8, the whole measurement routinebegins again. That way, the sample 8 can be scanned and measuredthree-dimensionally.

We claim:
 1. A device for optically measuring a point of a sample withhigh local resolution comprising:a light source to emit an excitinglight beam suitable for exciting an energy state in the sample; a lensfor focusing the exciting light beam on the point of the sample that canbe arranged in a focal range of the lens; a separation device forseparating out the emission light spontaneously emitted by the samplebased on the excitation of the energy state; and a detector to detectthe emission light, wherein a stimulating light beam coming from thelight source to produce stimulated emission of the sample excited by theexciting light beam in the point of the sample, and wherein the excitinglight beam and the stimulating light beam are arranged so that theirintensity distributions partially overlap in the focal range.
 2. Thedevice according to claim 1 wherein the sample is arranged on apositioning table, with which a mechanical raster movement can becarried out at least in the direction of the optical axis (A).
 3. Thedevice according to claim 1 wherein between the light source and thelens, there is a beam-raster device for controlled scanning of thesample with the exciting light beam and the stimulating light beam. 4.The device according to claim 1 wherein the stimulating light beam islaterally staggered in relation to the exciting light beam in the focalplane.
 5. The device according to claim 2 wherein the stimulating lightbeam is staggered along the optical axis in relation to the excitinglight beam.
 6. The device according to claim 1 wherein at least oneother stimulating light beam coming from the light source is provided,whose intensity distribution in the focal range of the lens is differentfrom the intensity distribution of the other stimulating light beams. 7.The device according to claim 6 wherein the stimulating light beam isspatially arranged symmetrically in relation to the exciting light beam.8. The device according to claim 1 wherein the light source includes alaser that emits portions of light of different wavelengths.
 9. Thedevice according to claim 1 wherein the light source includes at leasttwo lasers that emit light of different wavelengths.
 10. The deviceaccording to claim 1 wherein at least one continuous-wave laser isprovided that emits the exciting light.
 11. The device according toclaim 1 wherein at least one laser is provided that emits light pulsesin a time sequence.
 12. The device according to claim 11, wherein alaser, which emits light pulses in a time sequence, produces stimulatinglight.
 13. The device according to claim 1 wherein the laser orproducing the stimulating light emits a light beam with a Gaussianprofile.
 14. The device according to claim 1 wherein the light sourcefor producing the stimulated emission is high intensity, so that thereis a nonlinear connection between that intensity and the occupation ofthe energy state of the sample.
 15. The device according to claim 1wherein the separation device includes a time-control device, with whichthe detector can be turned on after a pulse of the stimulating lightdies.
 16. The device according to claim 1 wherein the separation deviceincludes a first polarization element connected on the input side of thelens to polarize the stimulating light and a second polarization elementconnected on the output side of the lens to polarize the light going tothe detector with a conducting direction orthogonal to the firstpolarization element connected to the input side of the lens.
 17. Thedevice according to claim 1 wherein the separation device has at leastone wavelength filter.
 18. The device according to claim 1 wherein theseparating device has a dichroitic mirror.
 19. The device according toclaim 1 wherein the detector is a point detector, which is arranged in aplane optically conjugated to the focal plane of the lens.
 20. Thedevice according to claim 1 wherein a focusing element and a filter areconnected to the detector on the input side, and wherein the filter isarranged in a plane optically conjugated to the focal plane of the lens.21. The device according to claim 1 wherein between the light source andthe lens there is a filter element that is permeable for wavelengths ofthe stimulating light, which has a nonpermeable central area and apermeable outer area for the wavelengths of the exciting light.
 22. Amethod for optically measuring a point on a sample with high localresolution, in which an exciting light beam is focused on the point tobe measured by means of a lens and there excites the energy state, andin which the emission light spontaneously emitted by the point based onthe excitation of the energy state is separated out and detected,characterized by the fact that the sample excited by the exciting lightbeam into the point is induced by a stimulating light beam to stimulatedemission, wherein intensity distributions of the exciting light beam andthe stimulating light beam partially overlap in the focal range of thelens.
 23. The method according to, claim 22 wherein the sample isscanned with the exciting light beam and the stimulating light beam.